玉米穀粒α- 澱粉?之純化與定性

Abstract

利用澱粉薄層技術觀察萌芽的玉米種子，發現澱粉水解活性最早出現在內子葉內側與胚乳交界處的上皮細胞(epithelium)，糊粉層在第二天之後也逐漸?生澱粉水解?；隨著幼芽之生長，糊粉層在胚乳的澱粉降解上漸趨重要。在萌發的種子中，α-澱粉?的活性佔總澱粉水解活性的70-80％。整個萌芽期間，玉米穀粒出現了五個澱粉水解?，Amyl-1,2,3,4,5,其中Amyl-5是β-澱粉?。 自內子葉及胚乳（糊粉層和澱粉質胚乳）中各提純了兩個α-澱粉?，分別命名為P-I, P-II和E-I, E-II; P-I與E-I對應於Amyl-3, P-II與E-II則是Amyl-4。由定性分析之結果觀察，P-I與E-I具有極相同的性質，而P-II則與E-II相似。(i)P-I和E-I的分子量均為49KD，且在膠體電泳時表現相同的泳動率；P-II和E-II的分子量則同為51KD。(ii)P-I和E-I的最適反應pH範圍為5.5-6.5; P-II和E-II則是pH6.0-7.0。(iii)此四個α-澱粉?的反應最適溫度皆為60℃，但P-II和E-II的酵素活化能(Ea)，溫度係數(Q10)及耐熱性分別比P-I和E-I略高一點。P-I和E-I的Ea為7.35，5,91 Kcal/mole，Q10為1.41，1.27；P-II和E-II的Ea則是11.46，10.32 Kcal/mole，Q10為1.55。1.47。(iv)玉米α-澱粉?的活性表現並不需要鈣離子，因此也不受EGTA的抑制。(v)鈣離子具有保護α-澱粉?抗拒熱變性的作用；在鈣離子存在時，α-澱粉?經過70℃/十五分鐘的處理，還保有60-70％的活性；除去鈣離子後則完全喪失了抗熱性，若再添加鈣鹽，則可以穩定α-澱粉?分子而恢復其抗熱性。鍶離子可以部份取代鈣離子穩定α-澱粉?分子的效果。(vi)四種α-澱粉?的活性均受到HgCl2,pCMB的抑制，其中P-II, E-II所受到的抑制較P-I, E-I嚴重。(vii)免疫轉印的結果顯示，四個α-澱粉?與其所誘導的四組抗體血清彼此間有交叉反應，表示四個純化的α-澱粉?分子具有相似的抗原決定基。 對去胚種子分離所得到的糊粉層而言，合成及分泌α-澱粉?，並不一定要求外加GA，但是外加GA仍然有明顯的促進作用；反之，ABA則有明顯的抑制效果。而內子葉對於植物荷爾蒙處理的反應與糊粉層並不盡相同，雖然添加GA時會有像糊粉層一樣的促進效果，但是ABA的施予卻完全不抑制α-澱粉?的合成。顯然，在玉米種子之內子葉和糊粉層組織中，α-澱粉?基因表現的調控機制並不完全相同，此有待進一步的探討。

The endosperm of maize kernel contains a large amount of starch. Upon seed germination, amylolytic enzymes are synthesized and secreted into endosperm by scutellum and aleurone layer to hydrolyze starch. By using starch-film technique to detect the appearance of amylase in the germinating maize kernel, the sequential occurence of hydrolytic activity was demonstrated along the germination course. One day after the beginning of imbibition, The starch-digestive zone started to show at the interface between scutellum and starchy endosperm, where the epithelium of scutellum is. The amylolytic activity of aleurone layer followed a day later and spread throughout the whole endosperm four days after imbibition. During the germination period, about 70 to 80 % of total amylolytic activity was contributed by α-amylase. Five amylolytic isozymes, namely Amyl-1,2,3,4, and 5, were separated and identified on native polyacrylamid gel by activity staining after electrophoresis. Two major α-amylases were purified from each 5-day germinating scutellar and endosperm tissue, designated as P-I/P-II and E-I/E-II, respectively. P-I and E-I were equivalent to Amyl-3, P-II and E-II were equal to Amyl-4. The properties of P-I were very similar to that of E-I, and those of P-II and E-II were alike. (i) The molecular weight of P-I is the same as E-I of 49KD; P-II and E-II were 51KD. (ii) The optimal pH for P-I and E-1 was ranging from pH 5.5 to 6.5; P-II and E-II were optimal from pH 6.0 to pH 7.0. (iii) The optimal reaction temperature for all four enzyme preparations was 60℃; while the activation energy and heat tolerance of P-II and E-II were much higher than those of P-I and E-I. (iv) Calcium ion was not required for enzyme avtivities, because these amylase preparations were not inactivated by removing of Calcium ion. (v) α-amylases were rather heat resistant, and Calcium ion was required for heat tolerance of enzymes. In the presence of 10 mM of Calcium ion, about 70% of activity was retained after heating at 70℃ for 15 min. After the removal of Calcium ion by dialysis, α-amylases became sensitive to heating. And re-introduction of Calcium ion into Calcium-depleted enzyme preparation could readily restore the thermotolerance of enzymes. Besides, Strontium ion could partially replace Calcium ion. (vi) Addition of HgCl2 and pCMB caused the inactivation of all four ezynme-preparations, and P-II and E-II were more sensitive than P-I and E-I. (vii) Four antibodies were directed to against P-I, P-II, E-I and E-II, respectively. The positive cross-reactions among 4 enzyme fractions and 4 antibody preparations indicated that four enzyme molecules possessed the immunochemical similarity. Plant hormone, GA, was not essentially required for the expression of α-amylase activity in either aleurone and scutellum. Whereas, the addition of GA3 did stimulate the synthesis and secretion of α-amylase. The treatment of ABA reduced the α-amylase activity by inhibiting the protein synthesis of α-amylase in aleurone layer. On the other hand, ABA did not caused the reduction of newly synthesized α-amlase. This differential effects implied that the regulation of α-amylase expression inside scutellum might bedifferent from that in aleurone layer.